Understanding Free and Complexed Enzyme 

Mechanisms and Factors Contributing to 

Cell Wall Recalcitrance

Michael G. Resch

SIMB SBFC

Clearwater, FL

4/29/2014

NREL/PR-2700-60927

NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.
Acknowledgements

Task Leaders

Gregg Beckham

Michael Himmel

Steve Decker

TEM and SEM Imaging

Bryon Donohoe

Peter Ciesielski

Intern

Jennifer Nill

Clean Fractionation

Rui Katahira

Kellene McKinney

Cellulose Allomorphs and XRD 

Ashutosh Mittal

BAT Team

Techno Economic Analysis

Mary Biddy

Funding

Biomass Technologies Office (BETO) Office of Science
3

Economic Impact of Reducing Enzyme Loading

Humbird et al. 2011
4

Gilbert and Fontes, Ann. Rev. Biochem. 2010 

Lynd et al. MMR 2002

“free” cellulases

“Free” vs. Complexed Enzymes
5

Different physical mechanisms of denconstruction

“Free” Cellulases Cellulosomes

Resch, M.G. et al. (2013) Energy and Environmental Science
6

Do Free and complexed enzymes degrade cellulose synergistically?

Avicel conversion at 10 mg/g

Resch, M.G. et al. (2013) Energy and Environmental Science
7

Enzyme Accessibility is Different

“Free” Enzymes Cellulosomes

• Cellulases are evenly 

distributed

• Cellulosomes are near 

the cell walls

Possible factors limiting cellulosome 

activity on DA pretreated biomass 

• Accessibility

• Low koff compared to free cellulases

• Binding to lignin

Resch, M.G. et al. (2013) Energy and Environmental Science
Clean Fractionation Pretreatment

(Organosolve)
9

CEF

Solvent Temp (°C) Acid 

(mol/L) Lignin (%) Glucan (%) Xylan (%)

MIBK/Acetone/H2O 140 0.025 10.63 63.31 17.47

MIBK/Acetone/H2O 140 0.05 3.67 82.28 6.56

MIBK/Acetone/H2O 140 0.1 3.33 87.19 3.08

CEF Chemical Composition
10

How does Clean Fractionation affect biomass morphology?

140°C 0.1M

Katahira, R. et al (in review)
11

How does Clean Fractionation affect enzyme saccharification?

Resch, M. et al (in review)
12

Affect of PT Severity on Enzymatic Saccharification 

10 mg/g enzyme loading, 

72 hours

Resch, M. et al (in review)
13

Synergistic Combination of CTec2 and Cellulosomes

Resch, M. et al (in review)
14

Technoeconomic Results from CF Work

Mary Biddy (NREL)
15

SEM of Digested Cellulose Enriched Fraction 

Resch, M. et al (in review)
16

TEM of Digested Cellulose Enriched Fraction

Resch, M. et al (in review)
17

Model of Enzymatic Deconstruction Mechanisms

Ciesielski, P., Resch, M. et al (in review)
Cellulose Allomorphs
19

Allomorph EH Study Objectives

Isolate cellulose allomorphs with identical 

crystallinity.

How do complexed, free and a combination 

perform on cellulose I, II and III?

Does the crystallinity change over the course 

of digestion?

? How does cellulose type effect the hydrolysis 

mechanism with free, complexed and 

combination?
20

Crystal Structure Models
21

Cotton linters Allomorphs - XRD

10 20 30 40

2θ

Cellulose I 

Cel I; CI - 62

PASC CL; CI - 44

PASC CL; CI - 34

Amorphous - Ball Milled - 6h

A

10 15 20 25 30 35 40

2θ

Cellulose II

Cel II @ 145C; CI - 57

Cel II @ 70C; CI - 49

Cel II @ 25C; CI - 40

Amorphous - Ball milled 6h

B

10 15 20 25 30 35 40

2θ

Cellulose III

Cel III @ 130C; CI - 60

Cel III @ 25C; CI - 49

Cel III @ -33C; CI - 32

Amorphous - Ball milled 6h

C

Allomorph Crystallinity Index (CrI)

Cellulose I 62 44 34

Cellulose II 57 49 40

Cellulose III 60 49 32

Ashutosh Mittal
22

Enzymatic Hydrolysis of Cellulose Allomorphs
23

XRD – 50% EH with Ctec2 

10 15 20 25 30 35 40

Cellulose III - Ctec2

Cellulose III

Cel III Ctec2

10 15 20 25 30 35 40

Cellulose I - Ctec2

Cellulose I

Cel I Ctec2

10 15 20 25 30 35 40

Cellulose II - Ctec2

Cellulose II

Cel II Ctec2

No change in XRD of cellulose samples after 50% digestion with Ctec2

Ashutosh Mittal
24

XRD – 50% EH with Cellulosomes

10 15 20 25 30 35 40

Cellulose III - Cellulosomes

Cellulose III Cellulosomes

Cellulose III

10 15 20 25 30 35 40

Cellulose I - Cellulosomes

Cellulose I

Cellulose I Cellulosomes

10 15 20 25 30 35 40

Cellulose II - Cellulosomes

Cellulose II

Cellulose II Cellulosomes

XRD of cellulose II and III allomorphs change after 50% digestion with Cellulosomes

• Change in the intensity of XRD from various crystal planes

• Emergence of a new peak at 2θ = 24°

Do cellulosomes attack crystalline regions selectively?

Currently looking at the digestions of cellulose allomorphs with combination of Ctec2 

and Cellulosomes
25

I

Free Complexed

II

? ?

III

? ?
26

Conclusions

• “Free” enzymes and cellulosomes have distinct mechanisms in cellulose 

and biomass deconstruction

• “Free” enzymes and cellulosomes are synergistic 

• Biomass composition and enzyme accessibility to cellulose ends affect 

cellulosomes more than “free” enzymes

• Combining properties of “free” and multi-domain enzymes in 

commercial enzyme cocktails could lead to enzyme cost reductions
Acknowledgements

Task Leaders

Gregg Beckham

Michael Himmel

Steve Decker

TEM and SEM Imaging

Bryon Donohoe

Peter Ciesielski

Intern

Jennifer Nill

Clean Fractionation

Rui Katahira

Kellene McKinney

Cellulose Allomorphs and XRD 

Ashutosh Mittal

BAT Team

Techno Economic Analysis

Mary Biddy

Funding

Biomass Technologies Office (BETO) Office of Science
28

Clean Fractionation – Xylan Conversion

Resch, M. et al (in review)